Fibrosis is an important secondary manifestation of inflammatory disorders that may nevertheless be the main source of organ dysfunction. Thus, in Crohns disease, fibrosis resulting from chronic intestinal inflammation is the cause of intestinal obstruction and fistula formation. Fibrosis and related connective tissue abnormalities may also be a primary disease manifestation. This is the case in systemic sclerosis (SSc/scleroderma), a complex disorder of connective tissue characterized by accumulation of extra-cellular matrix in the skin and various internal organs. Recently it has been shown that down-regulation of Friend leukemia integration-1 (Fli1) as well as Kruppel-Like Factor 5 (KLF5) in fibroblasts via hypermethylation recapitulates all of the features of SSc. However, the mechanism underlying the relation of such down-regulation to fibrosis is not yet defined and, in addition, it is not known if and how Fli1 and/or KLF5 influence secondary fibrosis such as that occurring in Crohns disease. In initial studies we conducted an extensive analysis of Fli1 regulation of fibrosis in Fli1 KO fibroblasts generated by CRISPR/CAS9 system. In performing Cas9 gene editing we chose a paired guideRNA/nickase system to reduce off-target effect and to enhance gene editing specificity and fidelity. Accordingly, paired gRNAs targeting Fli1 (designed by the ZiFiT program) along with a Csy4-nickase Cas9 cassette were mounted in one lentiviral vector by modifying pLenti-CRISPRv2. The latter was then transfected into NIH 3T3 cells and colonies with vector integration were selected by culture with puromycin. Confirmation of the efficiency of gene knockout in selected colonies was determined by T7 assay, sequencing, and Western blot analysis. With these Fli1-deficient cells in hand, we evaluated collagen and profibrotic cytokine production in the absence of Fli1. To this end, we measured altered function of Fli1 KO cells (compared to WT cells) at the mRNA level using RNA extracted from cultured cells, reverse-transcribed and then quantitated with real-time PCR using Taqman Gene Expression assays. In multiple studies we conducted assays of Col1a1, Col1a2, Tgfb1, CTGF, Il6, Fn1, Il11, Igf1, Igfbp3, Igfbp5, Csf2 mRNA production of cells cultured alone or in the presence of various antibodies and inhibitors. The latter included anti-TGF-1,2,3 Ab (1D11), TGF-1 receptor (ALK5) inhibitor SB-431542, anti-mouse IL-6 Ab and IL-6R Ab, Anti-IGF1-R Ab, mouse rIL-11, Erk1/2 inhibitor U0126, PI3K inhibitor LY294002, dual PI3K/mTOR inhibitor BEZ235, GSK-3 inhibitor SB216763, mTORC1/2 inhibitor PP242, and BET bromodomain inhibitor JQ1. We found that Fli1 KO fibroblasts exhibited numerous positive and negative changes in function compared to WT cells. Most notably this included a 3-5 folds increase in Col1a1 and Col1a2 synthesis that was indicative of previously reported Fli-1 inhibition of collagen synthesis. In addition it included increased synthesis of Tgf1, IL-6, Fn1, Igf1, and Csf2. Conversely, it included decreased IL-11, Igfbp3, and Igfbp5 synthesis. Thus, antibody neutralization of TGF-1, IL-6/IL6R, IGF-1, singly or in combination failed to suppress increased collagen production by Fli1KO cells; in addition, autocrine production of TGF-1 or IL-6 by these cells was not the main mechanism for increased synthesis of collagen or other extracellular matrix components by these cells. A very surprising and potentially important finding was that both Col1a1 and Col1a2 mRNA synthesis by Fli1 KO cells was dramatically inhibited by a PI3K inhibitor, LY294002, strongly suggesting that a PI3K-Akt signaling pathway was regulating collagen synthesis in KO cells. Since LY294002 is also an inhibitor of bromodomain-containing protein 4 (BRD4), in further studies we determined if a specific inhibitor of BRD4, JQ1, also inhibited collagen synthesis in Fli1 KO cells. we found that, indeed, JQ1 also inhibited collagen synthesis in Fli1 KO cells and that JQ1 in combination with a dual inhibitor of PI3K/mTOR, BEZ235, caused virtually complete inhibition of collagen synthesis in these cells. An additional finding is that BRD4 production is increased 4-fold in Fli-1 deficient cells. The discovery that PI3K and BRD4 are important regulators of collagen synthesis in the face of Fli1-deficiency opened a new avenue of research into the mechanism of Fli1 function and its regulation of fibrosis in fibrotic diseases. In current studies we determined that Gli-2 signaling, a component of the Hedgehog signaling pathway, is activated in Fli1-deficient fibroblasts and that inhibition of GLi-2 signaling by GANT61, causes down-regulation of increased collagen synthesis by the deficient cells. In addition, we found that Fli1 deficient cells secrete a factor that interacts with a fibroblast surface receptor to initiate PI3K signaling. Thus, it became apparent that Fli1 deficiency acts via GLi-2 to induce a factor that, in turn, induces collagen synthesis via PI3K. The fact that enhanced collagen production by Fli1-/- cells is dependent on PI3K signaling suggested that these cells are being auto-stimulated via a receptor responding to a secreted ligand or via a receptor that is atypically hyper-responsive to a factor normally present in the culture medium with respect to collagen production. In initial studies to investigate this question we focused on IRS-1/IRS-2, adaptor proteins that mediates PI3K signaling due to stimulation cells by insulin and IGFs as well as by cytokines and various growth factors since these adaptor proteins are targets of Gli proteins. We found that IRS-1/IRS-2 is greatly upregulated in Fli1-/- cells as compared to wild type cells and that an inhibitor of IRS-1/2 (NT157) caused virtually complete inhibition of collagen production. Since cytokines were unlikely to be produced in the fibroblast cultures and inhibition of Nintedinib had only mild effects on collagen production this result suggested that either IGF or insulin receptors were being stimulated in Fli1-/- cells to induce PI3K. On this basis we explored the possibility that Fli1-/- cells have atypical responses to insulin signaling via its insulin receptor and IRS. To investigate this possibility we treated Fli1-/- cells with InsR siRNA or with anti-Insulin. and found that InsR siRNA caused substantial reduction in collagen production. More importantly, addition of anti-Insulin to the cell culture caused virtually complete loss of collagen production by Fli1-/- cells in the absence of increased cell death. This strongly suggested that the source of PI3K signaling and down-stream induction of collagen in Fli1-/- cells was via atypical insulin stimulation of the conventional insulin receptor. Finally, we investigated the molecular basis of Fli1 regulation of collagen synthesis and its relation to PI-3K. In previous studies it have provided some evidence that Fli1 deficiency facilitates increased collagen production by allowing binding of Ets-1 to sites otherwise occupied by Fli1. To examine this possibility in Fli1-/- cells we conducted ChIP-seq studies of these cells and WT cells and found that Ets1 binding was increased in the promoter regions of both Col1A1 and Col1A2 in the Fli1-deficient cells as compared to WT cells. The same pattern was observed with respect to the Gli2 (but not to the Gli1) promoter and the IRS-1 (but not the IRS-2) promoter). These findings were amplified by promoter-luciferase studies that showed that Ets-1 binding to the TSS-2 start site of the Gli 2 promoter is important for Gli2 expression and in turn depends on co-binding with Sp1. The direct relevance of PI3K to to such binding is shown by the fact that inhibition of PI3K signaling greatly diminishes Ets1 bind in ChIP studies.